Regulation of human monocyte adherence by granulocyte-macrophage colony-stimulating factor.

Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to increase the adherence of purified peripheral blood monocytes to plastic surfaces and to monolayers of human umbilical vein endothelial cells. With plastic surfaces as a model 9-hr culture with GM-CSF was necessary for enhancement, and maximum levels were obtained after 24-hr stimulation. GM-CSF-stimulated adherence must require new RNA and protein synthesis because actinomycin D and cycloheximide abolished existing adherence and prevented further monocyte attachment. Interestingly, shorter incubations (1-2 hr) with cycloheximide increased adherence, suggesting a labile inhibitor. Formaldehyde fixation of monocytes but not of human vein endothelial cells abolished adherence, indicating the need for actively metabolizing monocytes. Thus, a hemopoietic growth factor, responsible for the proliferation and differentiation of monocytes, can also alter their adhesive characteristics. These observations may have important implications in pathological situations and in the in vivo use of GM-CSF.     

In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patients with normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; Sandoz/Schering-Plough, 5.5 micrograms protein/kg/d, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) in S phase (evaluated by the suicide technique with high 3H-Tdr doses) increased from 31% +/- 16% to 88% +/- 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage I-III forms. Moreover, by autoradiography (after incubation of marrow cells with 125I-labeled GM-CSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitors was not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSF-induced proliferative stimulus into enhanced platelet production.     

Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures.

The ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM-CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM-CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo.     

Outcomes of granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor use in neutropenic patients infected with human immunodeficiency virus.

OBJECTIVE: To characterize the effects of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) on clinical outcomes in neutropenic HIV-infected patients, by means of a retrospective cohort study at an urban teaching hospital. METHOD: Data were reviewed from all patients discharged between January 1, 1996, and August 31, 1997, with human immunodeficiency virus and neutropenia (absolute neutrophil count (ANC) <1000 cells/mL), with outcome measures of length of stay, infectious complications, and survival to discharge. RESULTS: Of the 228 discharged patients who met selection criteria, 71 had received G-CSF or GM-CSF; 157 controls had not. Cases had lower CD4+ cell counts (30 vs. 54 cells/mL; P = 0. 017) and lower nadir ANCs (372 vs. 579 cells/mL; P < 0.001). Granulocyte-CSF or GM-CSF usage was not associated with the frequency of site-related infections, fever, or sepsis (all P > 0. 20). No difference was found in duration of hospitalization (23 vs. 21 days; P > 0.20). In a logistic regression model for survival to discharge, higher nadir ANC and CSF use were independently associated with improved survival (P = 0.034 and P = 0.026, respectively). CONCLUSION: Use of G-CSF or GM-CSF was associated with improved survival to discharge among hospitalized HIV-infected patients with neutropenia.     

A pilot study of low-dose recombinant human granulocyte-macrophage colony-stimulating factor in chronic neutropenia

Summary. Recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) is under investigation for the treatment of a wide range of haematological disorders. At commonly used doses of > 120 μg/m²/d, extramedullary toxicity is common. We report the effects of low-dose (LD) rhGM-CSF in patients with chronic neutropenia related to HIV infection, myelodysplastic syndrome and idiopathic neutropenia. Nine patients with a mean pre-treatment neutrophil count of 0·6 × 10⁹/1 (range 0·2–1·4 × 10⁹/1) received daily rhGM-CSF at doses of between 5 and 15 μg/m², Eight patients responded with a mean post-treatment ANC of 3·2 × 10⁹/1 (range 1·9–4·6 × 10⁹/1). There was no significant therapy-related morbidity. We conclude that in chronic neutropenia, LD rhGM-CSF is an acceptable treatment which has important cost/benefit implications.     

The effect of recombinant human granulocyte-macrophage colony-stimulating factor on neutropenia and related morbidity in chronic severe neutropenia

STUDY OBJECTIVE: To define the clinical and hematologic effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on patients with chronic severe neutropenia. DESIGN: Open-label, phase II study of rhGM-CSF. SETTING: Inpatient hematology and surgery clinic at a university medical center. PATIENTS: Four consecutive patients with chronic severe neutropenia, which in two cases was complicated by severe infection, in one case by perianal fistula, and in one case by complete rectal prolapse. Two patients had chronic idiopathic neutropenia; one patient had congenital neutropenia (myelokathexis); and one patient had autoimmune neutropenia. INTERVENTIONS: The rhGM-CSF was given intravenously or subcutaneously at starting dosages of 150 to 1000 micrograms/m2 body surface area.d for 12 to 14 consecutive days. Two patients received a second course of daily rhGM-CSF treatment after a nontreatment interval of 14 to 20 days. MEASUREMENTS AND MAIN RESULTS: In all four patients, the absolute neutrophil counts increased from less than 0.25 x 10(9)/L to 3.2 to 19.2 x 10(9)/L within 2 weeks of beginning rhGM-CSF therapy. Two patients had life-threatening infections that resolved during therapy. The two other patients had major ano-rectal surgery during rhGM-CSF treatment and had no postoperative infections. CONCLUSIONS: In patients with chronic neutropenia, rhGM-CSF may increase neutrophil counts. This therapy may be a useful adjunct to antibiotic therapy for patients with infection and perioperatively for patients having anorectal surgery.     

Stimulation of myelopoiesis in chronic lymphocytic leukemia and in other lymphoproliferative disorders by recombinant human granulocyte-macrophage colony-stimulating factor

In an attempt to stimulate granulopoiesis, we administered recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) to 11 patients with lymphoproliferative disorders. Ten patients had neutropenia, six of whom had severe neutropenia (less than 500 neutrophils), including two with agranulocytosis. GM-CSF (60-250 micrograms/m2/day) was administered by continuous intravenous infusion daily for 14 days at 2-week intervals. Significant increases in white blood cell counts (1.3-to 11.7-fold) and neutrophils (1.7- to more than 29-fold) were seen in 10 of 11 patients, including one patient with agranulocytosis. Eosinophils (3.9- to greater than 65-fold) and monocytes (1.3- to 5-fold) increased as well. In contrast, no significant increases were seen in total lymphocytes or in different phenotypic subsets of lymphocytes during treatment. The overall proportion of myeloid and lymphoid elements in bone marrow remained stable. These results indicate that GM-CSF is effective in stimulating myelopoiesis in neutropenic states associated with lymphoproliferative disorders. Further studies will be necessary to determine whether the correction of neutropenia ultimately translates into clinical benefit.     

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.     

Outcomes of granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor use in neutropenic patients infected with human immunodeficiency virus

Objective: To characterize the effects of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) on clinical outcomes in neutropenic HIV-infected patients, by means of a retrospective cohort study at an urban teaching hospital.Method: Data were reviewed from all patients discharged between January 1, 1996, and August 31, 1997, with human immunodeficiency virus and neutropenia (absolute neutrophil count (ANC) <1000 cells/μL), with outcome measures of length of stay, infectious complications, and survival to discharge.Results: Of the 228 discharged patients who met selection criteria, 71 had received G-CSF or GM-CSF; 157 controls had not. Cases had lower CD4+ cell counts (30 vs. 54 cells/μL; P = 0.017) and lower nadir ANCs (372 vs. 579 cells/μL; P < 0.001). Granulocyte-CSF or GM-CSF usage was not associated with the frequency of site-related infections, fever, or sepsis (all P > 0.20). No difference was found in duration of hospitalization (23 vs. 21 days; P > 0.20). In a logistic regression model for survival to discharge, higher nadir ANC and CSF use were independently associated with improved survival (P = 0.034 and P = 0.026, respectively).Conclusion: Use of G-CSF or GM-CSF was associated with improved survival to discharge among hospitalized HIV-infected patients with neutropenia.     

Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in hematopoiesis and host defense via interaction with specific cell-surface receptors in target tissues. We identified a truncated, soluble form of the low-affinity GM-CSF receptor (GMR) in chorio-carcinoma cells. Low-affinity GMR cDNAs encoding both the membrane-bound and soluble receptors were obtained by PCR using primers corresponding to the published sequence. Clones encoding the soluble receptor were identical in sequence to the membrane-bound form but contained a 97-nucleotide internal deletion. The amino acid sequence of this deleted cDNA predicts a protein that lacks the 84 C-terminal amino acids of the membrane-bound receptor, including the transmembrane and cytoplasmic domains, and contains 16 different amino acids at its C terminus. Expression of the soluble GMR cDNA in murine psi-AM cells as well as GM-CSF-dependent myeloid 32Dc13 cells produced a secreted protein that retained its capacity to bind GM-CSF in solution. RNase protection analysis indicates that this variant cDNA is derived from a naturally occurring mRNA. Soluble receptors have been identified for several other hematopoietin receptors and may be a general feature of this class. The striking similarity between the soluble form of the GMR and other hematopoietin receptors suggests that soluble binding proteins may play an important role in regulating the broad spectrum of biological responses mediated by these cytokines.     

Augmentation of the immune response with granulocyte-macrophage colony-stimulating factor and other hematopoietic growth factors

Granulocyte-macrophage colony-stimulating factor is by far the most widely used hematopoietic growth factor to augment immune responses. At present, the best secured effect is as an adjuvant cytokine for vaccination. Granulocyte-macrophage colony-stimulating factor can be delivered as gene-transduced tumor cells, as plasmid DNA, or as the soluble free granulocyte-macrophage colony-stimulating factor protein. Granulocyte-macrophage colony-stimulating factor must be present at the same site as the vaccine component. Granulocyte-macrophage colony-stimulating factor may also augment the effect of therapeutic monoclonal antibodies by enhancing various effector functions such as antibody-dependent cellular cytotoxicity and amplifying an idiotypic network response (i.e., antitumor immunity). It may also be advantageous to combine granulocyte colony-stimulating factor with monoclonal antibodies (neutrophil and monocyte antibody-dependent cellular cytotoxicity) for tumor therapy. However, these growth factors might also induce immune suppression, which may hamper the contemplated effect of the growth factor. It is urgently warranted to better understand these dual effects on the immune system so that we can find optimal uses for the growth factors in various clinical settings.     

The human granulocyte-macrophage colony-stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% +/- 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% +/- 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.